Tetranectin is a Ca2+-binding trimeric C-type lectin which is present in blood plasma and from the extracellular matrix of certain tissues. The tetranectin group of proteins comprises tetranectin isolated from man and from mouse and the highly related C-type lectin homologues isolated from the cartilage of cattle (Neame and Boynton, database accession number PATCHX:u22298) and from reef shark (Neame et al., 1992, Neame et al., 1996 and database accession number p26258 and PIR2:A37289).
The mature tetranectin polypeptide chain of 181 amino acid residues is encoded in three exons as shown by molecular cloning and characterisation of the gene (Berglund & Petersen, 1992; Wewer & Albrechtsen, 1992). Exon 3 of the human tetranectin gene encodes a separate functional and structural unit, a single long-form so-called carbohydrate recognition domain (CRD), with three intra-chain disulphide bridges. The tetranectin CRD is considered to belong to a distinct class of C-type lectins (Day, 1994) clearly related to C-type lectins by sequence homology, conservation of disulphide topology (Fuhlendorff et al, 1987) and by the presence of an almost conserved suit of amino acid residues known to be involved in binding of calcium ions.
A published poster (Holtet et al 1996) has proposed tetranectin to be a trimer and that trimerisation is governed by the peptide encoded by exon 1. The peptide encoded by exon 1 was proposed to be “necessary and sufficient to govern trimerisation” whereas the polypeptide encoded by exon 2 was proposed as being “involved in lysine-sensitive binding to plasminogen”.
Tetranectin was first identified as a plasma protein binding to plasminogen by binding to the kringle-4 domain of plasminogen. Recent unpublished results (Graversen et al., manuscript for PNAS) proves (1) that the site in tetranectin involved in binding to plasminogen resides entirely in the CRD-domain (encoded by exon 3), (2) that binding is calcium sensitive, and (3) that the kringle-4 binding site in tetranectin overlaps the putative carbohydrate binding site of the CRD domain. Hence, there is now surprising definitive evidence that TN exons 1 and 2, i.e. the trimerisation unit in TN does not exhibit any plasminogen-binding affinity. Accordingly, an artificial protein containing a TTSE unit as part of its architecture is not expected to interact with plasminogen or plasmin due to properties inherited from tetranectin.
Tetranectin has also been reported to bind to sulfated polysaccharides like heparin (Clemmensen (1989) Scand J. Clin. Lab. Invest. vol 49:719-725). We have new results showing that the CRD domains of tetranectin are not involved in this protein-polysaccharide interaction. In fact, the site in tetranectin is located in the N-terminal region of exon 1 and may be abolished by removal or mutagenis of N-terminal lysine residues (Graversen et al., manuscript), processes that do not inhibit trimerisation. TTSEs that include most or all of TN exon 1 therefore confer an affinity for sulfated polysaccharides to any designed protein which encompasses such a TTSE as part of its structure. If desired, however, this affinity can be reduced or abolished by N-terminal truncation or mutagenesis of lysine residues in the part of the TTSE that corresponds to the N-terminal 8-10 amino acid residues of exon 1 (Graversen et al., unpublished). With respect to gene therapy which is also withing the scope of the present invention, there is only a limited number of basic strategies for gene therapy which show some promise in preclinical models so far. The two major strategies e.g for the treatment of malignant tumors are cytokine-gene aided tumor vaccination and selective prodrug activation. Whereas the first strategy relies on the strong immunostimulatory effect of a relatively small number of genetically modified cytotoxic T cells or tumor cells, the second one is based on conversion of a nontoxic prodrug into a toxic product by an enzyme-encoding gene where the toxic effect is exerted also on non-transduced dividing tumor cells due to a so-called bystander effect. Alternatively, strategies can be envisaged where the malignant phenotype of a cell is reversed by either inactivating an oncogene or reestablishing an inactivated tumor suppressor gene. In both cases, highly efficient gene transfer to the cells in a tumor is required. Although high efficiencies of gene transfer can be obtained in vitro and even in vivo under certain circumstances, correction of the malignant phenotype by reversing the major oncogenic change in the tumor cells is unlikely to result in normal cells. Thus, selective induction of tumor cell death by use of the present invention would be preferable, and the development of methods enabling such induction will be of great importance.
A major problem in connection with the gene therapy is the incorporation of foreign material into the genome. Viruses, however, have only been partially successful in overcoming this problem. Hence the initial efforts at gene therapy are still directed towards engineering viruses so that they could be used as vectors to carry therapeutic genes into patients. In the still very immature in vivo method of somatic gene therapy, where a vector could be injected directly into the bloodstream, or more preferably by transmucosal delivery, the present invention may be utilized due to the surprising number of ways the gene therapy may be targeted.
For many gene-therapy applications in the future, it is probable that a synthetic hybrid system will be used that incorporates engineered viral component for target-specific binding and core entry, immunosuppressive genes from various viruses and some mechanism that allows site-specific integration, perhaps utilizing AAV sequences or an engineered retroviral integrase protein. In addition, regulatory sequences from the target cell itself will be utilized to allow physiological control of expression of the inserted genes. All these components would be assembled in vitro in a liposome-like formulation with additional measures taken to reduce immunogenicity such as concealment by PEG
As mentioned, one of the current problems in gene therapy is the efficient delivery of nucleic acids to as many as possible of a specific population of cells in the body, and it is often not possible to find e.g. an appropriate viral vector that will find that particular cell population efficiently and selectively (Review on aspects of gene therapy: Schaper, W & Ito, W. D. Current Opinion in Biotechnology, 1996, vol. 7, 635-640. Nature Biotechnology 1998 vol 16 is an entire volume dedicated to protein- and gene delivery).
Given the possibility of in vitro generation of a human antibody against virtually any target antigen by phage technology, it follows that TTSEs, where one of the subunits is modified with a membrane integrating or associating entity, may be used as a practicable tool for generating a viral, bacterial or preferentially artificially assembled lipomal vehicle that will allow selective delivery of the contained material by infection or transfection of any cell population to which such a specific antibody may be generated. Moreover vehicles may, with the use of TTSEs, be individualised by selection of patient specific antibodies or by assembling TTSE units conjugated with scFvs selected from an ensemble of antibodies selected by the particular markers of the disease.